1. Field of the Invention
A PCR method is provided for detecting BK virus, and particularly for managing BK virus-associated nephropathy in renal transplant patients along with related PCR primers and primer sets. A method for reducing BK virus viral load also is provided.
2. Description of the Related Art
BK virus (BKV) is a human polyoma virus that was originally isolated from the urine of immunocompromised patients. Since then, a number of BKV variants (subtypes) have been isolated. BKV causes a subclinical (asymptomatic) infection in the majority of the general population within the first 10 years of life. Subsequent to infection, the virus normally remains latent in the kidney. However, the virus may become reactivated at a later point in time as a result of immunosuppression, for example, following renal transplantation.
BKV contains a double stranded DNA (dsDNA) genome. The complete DNA sequence of BKV is approximately 5,100 base pairs, however this varies with each variant of BKV. For example, the Dunlop strain of BKV contains 5,153 base pairs (see, for example, Self et al. (1979), xe2x80x9cThe Genome of Human Papovavirus BKV,xe2x80x9d Cell 18:963-77, incorporated herein by reference in its entirety). The BKV genome contains a coding region and a non-coding control region, but is functionally divided into three regions. The coding region can be further divided into the early region and the late region. The early region contains the coding sequence for two non-structural proteins: the T-antigen protein and the t-antigen protein. The late region contains the coding sequence for four structural proteins: VP-1, VP-2, VP-3 and the agno protein. The non-coding control region contains the transcriptional control elements for both early and late gene expression, as well as containing the viral origin of replication.
The role of BKV infection in renal allograft dysfunction has been controversial. Studies have reported a range of clinical outcomes, varying from asymptomatic to significant renal dysfunction. However, recent studies have shown that BKV causes nephropathy in up to 5% of renal allografts. In addition, BKV infection in renal transplant recipients can be associated with significant morbidity. The controversy surrounding the role of BKV in renal dysfunction is due to the difficulty in diagnosing and monitoring BKV infection. One problem is that serological and traditional viral culture techniques are either not specific or not readily available. In addition, these techniques may not be practical in situations where rapid diagnosis is required in order to make timely patient management decisions.
The diagnosis of BKV-associated nephropathy (BKVN) usually is made by allograft biopsy. A positive biopsy shows viral inclusion bodies, often associated with variable mononuclear infiltrates, and tubulitis that may resemble acute rejection. Immunohistochemical techniques are not sensitive enough to detect latent virus in biopsies that lack viral inclusion bodies that are otherwise detectable by standard light microscopy. A complicating factor in the diagnosis of BKVN nephropathy is the fact that BKVN can present in many different ways. BKV infection has presented with features that were variably diagnosed as acute rejection, interstitial nephritis, drug toxicity, ureteric stenosis, and also asymptomatic. Thus a great degree of caution and a high index of suspicion are needed for the proper diagnosis and management of BKVN.
Even after BKVN has been properly diagnosed, clinical management remains a significant challenge. No clinically proven anti-polyomavirus drugs are currently available for the management of BKVN even though in vitro studies have demonstrated that several drugs, including retinoic acid derivatives, DNA gyrase inhibitors, cytosine arabinoside and cidofovir, inhibit polyoma viral DNA replication. Although various therapeutic strategies have been suggested and tried, the results are often variable and dismal. As a result, therapy for BKVN is usually based on renal allograft biopsy findings. The difficulty in clinical management is compounded by the fact that even when biopsies show tubulitis, suggesting the possibility of underlying rejection, there is little or no transient response to corticosteroids in most cases. Another possibility for clinical management is to reduce immunosuppression. Although reducing immunosuppression decreases the viral load, it increases the risk of rejection.
Given the challenges posed by the diagnosis and management of BKVN, the development of non-invasive quantitative techniques to monitor viral load can have a significant impact on the clinical management of these cases. For example, quantitation of the viral load would allow a physician to monitor a patient""s response to specific anti-viral therapy. In addition, it would allow a physician to lower a patient""s level of immunosuppression sufficiently to permit stimulation of anti-viral immunity without reducing it to a point that would precipitate acute rejection.
Several references discuss PCR assays for the detection of BKV in urine and/or blood. However, those references teach primers that bind to and amplify BKV viral DNA in the early (T and t antigen) region. Other references disclose PCR primers that bind to and amplify sequences located in the late region of the BKV genome, and particularly in the VP-1 region (Li Jin, xe2x80x9cMolecular Methods for Identification and Genotyping of BK Virus,xe2x80x9d Methods in Molecular Biology, vol. 165, pp. 33-48 (2001), Li Jin, xe2x80x9cRapid Genomic Typing of BK Virus Directly from Clinical Specimens,xe2x80x9d Molecular and Cellular Probes, vol. 7, pp. 331-334 (1993), Li Jin, xe2x80x9cGenomic Typing of BK Virus in Clinical Specimens by Direct Sequencing of Polymerase Chain Reaction Products,xe2x80x9d J. Medical Virology, vol. 41, pp. 11-17 (1993), and Baksh et al., xe2x80x9cMolecular Genotyping of BK and JC Viruses in Human Polyomavirus-Associated Interstitial Nephritis After Renal Transplantation,xe2x80x9d Amer. J. Kidney Disease, vol. 38, no. 2, pp. 354-365 (August 2001) each of which are incorporated herein by reference in their entirety). These primers function to generate PCR amplicons that were used to distinguish BKV subtypes by PCR combined with restriction enzyme analysis or sequencing analysis.
Cidofovir (HPMPC, Vistide, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine) is an acyclic nucleoside phosphonate with broad-spectrum activity against a wide variety of DNA viruses. It is reportedly effective in patients diagnosed with progressive multifocal leukoencephalopathy, an infection caused by JC virus. Cidofovir is usually administered in a dosage of 5 mg/kg, with approximately 75-80% of the cidofovir dose excreted in the urine unchanged within 24 hours of administration. In addition, cidofovir is usually given in conjunction with the anti-diuretic probenecid. Cidofovir is nephrotoxic and, therefore, is contraindicated in patients with impaired renal function. Nevertheless, cidofovir was studied herein for its effect on BKV load in kidney transplant recipients diagnosed with BKV associated nephropathy.
Provided are PCR primers for the quantitation of BKV viral loads in a sample using a PCR or quantitative PCR (QPCR) assay and methods for the diagnosis and management of BKVN. PCR primers are provided for binding to and amplifying a region of the BKV genome using PCR or QPCR. The PCR primers include a forward BKVN primer, preferably including substantially the sequence 5xe2x80x2-TGATAGCCCAGAGAGAAAAATGC-3xe2x80x2 (SEQ ID NO: 1), and/or a derivative thereof, and a reverse BKVN primer, preferably including substantially the sequence 5xe2x80x2-TCCACAGGTTAGGTCCTCATTTAAA-3xe2x80x2 (SEQ ID NO: 2), or a derivative thereof.
Also provided is a method for quantitating the BKV viral load in a viruria, serum or plasma (viremia) tissue sample using one or more of the BKNV primers, or derivatives thereof, in a PCR or QPCR assay. The method includes the steps of: performing a nucleic acid purification method on a patient specimen to obtain a nucleic acid test sample; conducting PCR or QPCR on the test sample using an appropriate primer set, as defined herein, to generate an amplicon if BKV target sequences are present in the test sample; and detecting the presence of BKV specific amplicon in the sample. A multiplexed QPCR method also is provided using the above-described primer set in combination with a second PCR primer set.
A method also is provided for the management of BKVN in renal transplant recipients using cidofovir, preferably without co-administration of an anti-diuretic such as probenecid. The method includes quantitating the BKV viral load in a patient specimen and administering to a patient a dosage of cidofovir effective to reduce BKV viral load in the patient. Patients treated according to the method have shown clearance of BKV viruria after 2-4 doses of the drug.
Also provided is a kit containing a PCR primer, or a derivative thereof, for quantitating BKV viral load in a sample using a PCR assay, for use in diagnosing and managing BKVN. The kit may further comprise, without limitation control nucleic acids, primer sets to generate a second amplicon in a multiplexed PCR or QPCR assay; collection containers; a buffer; nucleic acid purification reagents or kits; a mixture of dNTP""s; and a thermostable DNA polymerase.